重组人内向整流钾离子（KCNJ）通道在酿酒酵母中的表达与纯化
Expression and purification of recombinant human inward rectifier K+ (KCNJ) channels in Saccharomyces cerevisiae.
作者信息
D'Avanzo Nazzareno, Cheng Wayland W L, Xia Xiaobing, Dong Liang, Savitsky Pavel, Nichols Colin G, Doyle Declan A
机构信息
Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
出版信息
Protein Expr Purif. 2010 May;71(1):115-21. doi: 10.1016/j.pep.2010.01.010. Epub 2010 Jan 11.
The inward rectifier family of potassium (KCNJ) channels regulate vital cellular processes including cell volume, electrical excitability, and insulin secretion. Dysfunction of different isoforms have been linked to numerous diseases including Bartter's, Andersen-Tawil, Smith-Magenis Syndromes, Type II diabetes mellitus, and epilepsy, making them important targets for therapeutic intervention. Using a family-based approach, we succeeded in expressing 10 of 11 human KCNJ channels tested in Saccharomyces cerevisiae. GFP-fusion proteins showed that these channels traffic correctly to the plasma-membrane suggesting that the protein is functional. A 2-step purification process can be used to purify the KCNJ channels to >95% purity in a mono-dispersed form. After incorporation into liposomes, (86)Rb(+) flux assays confirm the functionality of the purified proteins as inward rectifier potassium channels.
钾离子内向整流通道家族（KCNJ）调节包括细胞体积、电兴奋性和胰岛素分泌在内的重要细胞过程。不同亚型的功能障碍与多种疾病相关，包括巴特综合征、安德森-陶威尔综合征、史密斯-马吉尼斯综合征、II型糖尿病和癫痫，这使得它们成为治疗干预的重要靶点。采用基于家族的方法，我们成功地在酿酒酵母中表达了所测试的11种人类KCNJ通道中的10种。绿色荧光蛋白融合蛋白表明这些通道能够正确转运至质膜，这表明该蛋白具有功能。两步纯化过程可用于将KCNJ通道以单分散形式纯化至纯度>95%。掺入脂质体后，铷-86通量测定证实了纯化蛋白作为内向整流钾通道的功能。
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